This application addresses broad Challenge Area (08) Genomics, and specific Challenge Topic 08-CA-104: Regulatory functions of small RNAs. Our goal is to identify small RNAs (smRNAs) as regulators of histone modifying enzymes, mediating interactions with promoters and/or non-coding RNAs present in promoter proximal regions and mediating long-term effects on gene expression. This is a novel model that would tie together genome-encoded small RNAs, non-coding RNAs, and the regulation of histone modifications, leading to long-term modulation of gene expression. Epigenetic modifications are inheritable alterations to the genome in the form of enzymatic manipulations of key histone residues, or the methylation of specific cytosines in the DNA sequence. Carcinogenesis is likely to include aberrant use of this pathway to cause long-term changes in gene expression in transformed cells. In fact, epigenetic dysregulation is a hallmark of numerous cancers, and compounds which broadly affect histone modifications are under intense scrutiny as cancer therapies. While this strategy may be effective, we believe that it will be possible to therapeutically target epigenetic modulation of selected genes by exploiting the existing RNAi machinery, as demonstrated for several smRNAs and genes. This project aims to understand the relationship between small, non-coding RNA sequences and epigenetic histone modifications, and explore the mechanisms by which epigenome-modifying complexes may be recruited by small RNAs to their target DNA sequences. Comparison of smRNAs identified in human embryonic stem cells by deep sequencing with genomic alignment sites and previously published epigenomic data of key histone modifications has identified striking association between these two previously independent pathways. In this proposal we will test the hypothesis that smRNAs mediate the placement of epigenetic marks. Specifically, we will (1) identify and characterize classes of smRNA sequences associated with specific chromatin modifications in human ES Cells during neural differentiation;(2) bioinformatically predict as yet unobserved smRNA-mediated epigenetic effects;and (3) experimentally confirm the functional interaction between small non-coding RNAs and epigenetic modifications in hESC. Epigenetic marking is one of the next great questions in biology and medicine. We propose that endogenous smRNAs are the key link between histone modification mechanisms and individual genomic loci. If smRNAs do in fact direct epigenetic modifications, this may represent a novel mechanism by which cells are capable of transcriptional regulation. This will provide both additional therapeutic targets and enhanced understanding of disease etiologies for regions of altered histone modification during cancers. PUBLIC HEALTH RELEVANCE: Epigenetic dysregulation is a hallmark of several human disorders and numerous cancers. Histone modifications represent one mechanism of epigenetic regulation that directly affects the transcriptional availability of adjacent genomic elements. This project aims to identify the relationship between small, non-coding RNA sequences and epigenetic histone modifications, and explore the mechanisms by which epigenome modifying complexes may be recruited by small RNAs to their target genomic regions. The association of small RNAs with specific histone modifications provides understanding of disease etiologies and suggests mechanisms to transcriptionally regulate target genes in patients.